Functional Analysis of the Genes of Yeast Chromosome V by Genetic Footprinting-Reference-Cited by-同舟云学术

Functional Analysis of the Genes of Yeast Chromosome V by Genetic Footprinting

Published:1996-12-20 Issue:5295 Volume:274 Page:2069-2074
ISSN:0036-8075
Container-title:Science
language:en
Short-container-title:Science

Author:

Smith Victoria¹,Chou Karen N.¹,Lashkari Deval²,Botstein David²,Brown Patrick O.³

Affiliation:

1. V. Smith and K. N. Chou, Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.

2. D. Lashkari and D. Botstein, Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.

3. P. O. Brown, Howard Hughes Medical Institute and Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.

Abstract

Genetic footprinting was used to assess the phenotypic effects of Ty1 transposon insertions in 268 predicted genes of chromosome V of Saccharomyces cerevisiae. When seven selection protocols were used, Ty1 insertions in more than half the genes tested (157 of 268) were found to result in a detectable reduction in fitness. Results could not be obtained for fewer than 3 percent of the genes tested (7 of 268). Previously known mutant phenotypes were confirmed, and, for about 30 percent of the genes, new mutant phenotypes were identified.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

Reference28 articles.

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3. Genetic footprinting: a genomic strategy for determining a gene's function given its sequence.

4. Dietrich F., et al.., Nature, in press. In subsequent releases, additional telomeric sequences were added, making the full length of chromosome V 574,860 bp [updated in the Martinsried Institute for Protein Sequences (MIPS) Yeast Genome Database]. The number of predicted genes is derived from Dietrich et al. and from the MIPS database. Most of the possible additional and alternative open reading frames noted in the MIPS database were also covered by the primers used in this study. One to three independent gene-specific primers were used to analyze each gene. Primers were designed with the use of the program PRIMER (Whitehead Institute for Biomedical Research, Cambridge, MA) with a specified melting temperature of 69° to 73°C. Primers were synthesized by means of a 96-well array synthesizer [D. A. Lashkari, S. P. Hunicke-Smith, R. Norgren, R. W. Davis, T. Brennan, Proc. Natl. Acad. Sci. U.S.A.92, 7912 (1995)] and labeled at their 5′ end with 5-carboxyfluorescein (Applied Biosystems or Pharmacia). Approximately 85% of primers produced usable data. Alternative primers were synthesized for any gene for which the first primer failed to produce usable data. Each labeled gene-specific primer was used in a PCR with an unlabeled Ty1-specific primer, as described (2).

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